Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a ...

Vamseedhar Rayaprolu

Montana State University

Hi,

Sorry for the delayed response. Please look at the original reference (I posted the link in the previous post). Also look at references 51-54 in that link. This method was developed by our collaborators in SF9 using a baculoviral expression system. Let me know if you need more information.

2016-03-05 11:16:06

Kathrin Schlicht

Universität Bielefeld

Hi Vamseedhar,

we produce our AAVs by calcium phosphate-based transfection of plasmid DNA into HEK cells. The viral titer we reach is bellow the titer you need to get a protein concentration that high even after concentration by a molecular cut off filter. So we can`t perform DSF. We really like to test this method. Maybe you know how many HEK cells were transfected.

Thank you for your quick response.

2016-03-02 23:42:39

Vamseedhar Rayaprolu

Montana State University

Hi Kathrin,

We determined concentrations using gel densitometry. You could also use UV 280 to determine the protein concentration but because our virus sample also had DNA, we preferred running the gel and doing the densitometry. You can increase your protein concentration using molecular a weight cut off filter though AAV samples tend to stick to the filters quite a bit. The samples were obtained from our collaborators. For more information on purification of the virus and checking integrity, please refer to http://jvi.asm.org/content/87/24/13150.full